Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in kappa-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a kappa-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in kappa-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.     

A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments

A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K₂PtBr₆ within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.     

Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

An enzyme catalyzing the conversion of μ- to κ-carrageenan has been demonstrated in both haploid and diploid plants of Chondrus crispus. It acts at the polymer level producing 3,6-anhydro-d-galactose with the stoichiometric release of sulfate. Two-thirds of the recoverable enzyme was associated with the 15,000g pellet most of which could be solubilized by passage through a Ribi Cell Fractionator. The enzyme precipitated between 2.65 and 4.24 m (NH₄)₂SO₄ and was partly purified on DEAE-cellulose columns. This sulfohydrolase has a pH optimum near 6.5 and is inhibited by molybdate, phosphate, sulfate, tungstate, cysteine, ATP, GTP, UDP, and by λ-carrageenan. No activator was found. The enzyme showed a similar affinity for several preparations of μ-carrageenan and for the κ-carrageenase-resistant fraction from κ-carrageenan thus confirming that the latter is a biosynthetically unfinished molecule.     

Effect of initial biomass concentration on the growth of immobilized Nitrosomonas europaea

Nitrosomonas europaea was immobilized in carrageenan-gel beads with a low and a high biomass concentration (100-fold difference). Under growth conditions in a continuous air-lift loop reactor, a low initial concentration yielded a few large micro-colonies per bead, whereas a high initial biomass concentration resulted in many small micro-colonies per bead. The macroscopic consumption rate of the latter was three times higher under the circumstances applied. This is also predicted by our colony-expansion model when diffusion limitation over the micro-colonies is incorporated. This model also predicts that diffusion limitation over the ultimate colonies is negligible when the initial biomass concentration exceeds 0.5 kg·m^–3 of carrageenan gel.     

Performance of growing Nitrosomonas europaea cells immobilized in κ-carrageenan

Summary Nitrosomonas europaea cells were immobilized in -carrageenan. The performance of the immobilized cells was investigated in an airlift loop reactor under wash-out conditions with respect to freely suspended cells. When fed with solutions of ammonia up to 16 mM, high substrate conversions were accomplished. Sudden increases in ammonium-ion concentration hardly influenced the conversion. Observations made by scanning electron microscopy showed that the immobilized cells were initially growing homogeneously across the beads, but as growth proceeded, a biomass density gradient developed, eventually resulting in a kind of biofilm.Offprint requests to: R. H. Wijffels     

Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O₂, NO₂⁻, and NO₃⁻ profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Maintenance Energy Demand and Starvation Recovery Dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi Cultivated in a Retentostat with Complete Biomass Retention

Nitrosomonas europaea and Nitrobacter winogradskyi (strain “Engel”) were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25°C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 μmol of O₂ mg⁻¹ of biomass-C · h⁻¹ for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 μg · ml⁻¹) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514–1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

Properties of a new strain of Nitrosomonas isolated from an aerobic biofilm in a domestic sewage-treatment system

A new ammonia-oxidizing strain, isolated from an aerobic biofilm in a domestic sewage-treatment system, was identified as a species of Nitrosomonas different from Nitrosomonas europaea. This strain had morphological features and growth characteristics typical of members of the genus Nitrosomonas. The G+C content of the DNA of this strain was 48.5 mol%, being lower than that of known strains of N. europaea. The extent of the homology between the DNA of this strain and that of other strains of N. europaea was less than 30%. After cells of this isolate, immobilized in a polyacrylamide gel, had been added to the aerobic reactor of a laboratory-scale sewage-treatment system, the concentration of ammonium nitrogen in the effluent decreased to 2 mg/l without the accumulation of nitrite, and removal of more than 70% of the nitrogen from the input sewage was achieved.     

Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log₁₀ CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 μg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12°C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Distribution of Nitrosomonas europaea and Paracoccus denitrificans Immobilized in Tubular Polymeric Gel for Nitrogen Removal

To improve the cooperative removal of nitrogen by Nitrosomonas europaea and Paracoccus denitrificans, we controlled their distribution in a tubular gel. When ethanol was supplied inside the tubular gel as an electron donor, their distributions overlapped in the external region of the gel. By changing the electron donor from ethanol to gaseous hydrogen, the distribution of P. denitrificans shifted to the inside of the tube and was separated from that of N. europaea. The separation resulted in an increase of the oxidation rate of ammonia by 25%.     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed V_λ and V_κ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.     

Microscale Vertical Profiles of N2 Fixation, Photosynthesis, O2, Chlorophyll a, and Light in a Cyanobacterial Assemblage

Profiles of ¹⁵N₂ fixation, O₂ production (gross photosynthesis), O₂ concentration, chlorophyll a concentration, and photon fluence rates were measured with 50-μm resolution in colonies of the heterocyst-forming cyanobacterium Nostoc parmelioides. Microelectrode measurements were made after 20 h of incubation under ¹⁵N₂ gas. Colonies were frozen, and 50-μm sections were prepared by using a freezing microtome and analyzed for ¹⁵N enrichment and chlorophyll a concentration. Colonies exhibited steep spatial gradients in rates of gross photosynthesis, O₂ concentration, and irradiance, with the highest values generally occurring at the surface. O₂ concentration, photosynthesis, and irradiance all showed positive correlations, but chlorophyll a concentrations varied independently of photosynthesis and irradiance. Forty-four percent of the variation in ¹⁵N incorporation was explained by gross photosynthesis (a positive correlation) when incorporation of ¹⁵N was expressed per unit of biomass (chlorophyll a).     

Na+ and K+ transport in Nitrosomonas europaea and Nitrobacter agilis

Potassium-depleted cells of Nitrosomonas europaea and Nitrobacter agilis were prepared by diethanolamine treatment and contained less than 5 mM intracellular K⁺. The addition of K⁺ to K⁺-depleted cells of N. europaea and N. agilis resulted in a depolarization of membrane potential (ΔΨ) by about 5 and 10 mV, respectively. This depolarization was, however, compensated by an equivalent increase in transmembrane pH gradient (ΔpH), so that the total proton-motive force (Δp) remained constant, indicating that K⁺ transport was electrogenic in both bacteria. Using ²²Na⁺-loaded cells, it is shown that both bacteria lack a respiration-dependent Na⁺ pump; however, antiporters for Na⁺/H⁺, K⁺/Na⁺ and K⁺/H⁺ were detected. Of these, at least the K⁺/Na⁺ antiporter required an electrochemical gradient for its operation. It is also shown that the unprotonated form of NH₄⁺ is transported into these bacteria by a simple diffusion mechanism.